Mass spectrometry histochemistry of peptides from formaldehyde-fixed, paraffin-embedded tissue

Abstract

The present invention in general relates to a method of localization and structural characterization of peptides in a formaldehyde-fixed paraffin-embedded biological sample using matrix assisted ionization (such as in MALDI). Specifically, the invention relates to the combination of the development of a sample preparation protocol which does not require any enzymatic digestion nor antigen-retrieval steps, with highly sensitive mass spectrometry.

The invention claimed is: 
     
       1. A method for imaging peptides in a formaldehyde-fixed paraffin-embedded biological sample, the method comprising:
 (a) providing a formaldehyde-fixed paraffin-embedded biological sample; 
 (b) sectioning the sample of (a); 
 (c) deparaffinizing the sectioned sample of (b); 
 (d) air drying the sectioned sample of (c); 
 (e) applying a MALDI (matrix-assisted laser desorption ionization imaging) matrix to the air-dried samples of (d), the MALDI matrix being selected from the group consisting of sinapinic acid, alpha-cyano sinapinic acid, cinnamic acid, alpha-4-cyano hydroxyl cinnamic acid, 2,5-dihydroxvbenzoic acid, 3-hydroxypicolinic acid, dithranol, derivatives thereof and combinations thereof; and 
 (f) performing a mass spectrometry analysis tuned for peptide analytes on the sample of (e); 
 
       wherein the method does not comprise enzymatic digestion, and wherein the method does not comprise antigen-retrieval, and wherein the method does not include use of a reactive MALDI matrix comprising 2,4-dinitrophenylhydrazine. 
     
     
       2. The method according to  claim 1 , wherein (c) is performed by immersing the paraffin-embedded sample 1-5 times in 100% xylene for about 1-5 min, followed by washing 1-5 times in absolute ethanol, each washing lasting about 1-5 min. 
     
     
       3. The method according to  claim 1 , wherein (c) is performed by immersing the sectioned samples of (b) sequentially in:
 (1) 100% xylene for about 3 min; 
 (2) 100% xylene for about 2 min; 
 (3) absolute ethanol for about 2 min; and 
 (4) absolute ethanol for about 1 min. 
 
     
     
       4. The method according to  claim 1 , wherein the MALDI matrix further comprises an additive to direct analyte ionization and desorption. 
     
     
       5. The method according to  claim 1 , wherein the mass spectrometry analysis is tuned for peptides. 
     
     
       6. The method according to  claim 4 , where the additive to direct analyte ionization and desorption is a Li +  salt. 
     
     
       7. The method according to  claim 1 , wherein the MALDI matrix is a MALDI matrix that does not promote liberation of aldehydes through reversal of aldehyde crosslinking or formaldehyde crosslinking.